Secondary cleavage of a fluorescently labeled SNAP-25 substrate by Xeomin ® drug product

Abstract

Objectives : To evaluate SNAP-25-type substrate cleavage product(s) generated by Botulinum neurotoxin Type-A (BoNT/A) and BoNT/A drug products utilizing a light-chain activity high-performance liquid chromatography (LCA-HPLC) method. Methods : Samples were reacted with a fluorescently labeled substrate derived from the SNAP-25 sequence. Reaction products were subsequently separated by reversed-phase HPLC. Cleavage products were chromatographically identified by elution position using fluorescence detection. Results : Xeomin ® drug product samples produced two fragments rather than one. Identification of the secondary fragment confirmed that secondary cleavage occurs at a position on the substrate corresponding to SNAP-25 Arg198–Ala199 as opposed to the expected BoNT/A cleavage site (corresponding to SNAP-25 Gln197–Arg198). This is also the cleavage site for Trypsin and Type-C toxin. The secondary cleavage was not observed in any BOTOX ® drug product samples, nor was it observed in 900 or 150 kDa bulk toxin samples under the experimental conditions employed. Only Xeomin ® drug product generated the additional unexpected cleavage product. All of the Xeomin ® lots analyzed exhibited this behavior. Conclusions : Possible explanations include a contaminating trypsin-like protease, concurrent Type-C expression behavior, and/or damage to the 150 kDa toxin causing non-specific cleavage uncharacteristic of pristine Type-A toxin.